This invention relates to the study of enzymatic events in cells and, more particularly, to the study of enzymatic events in single cells by optical detection methods. This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).
Enzymatic events in cells include steroid biosynthesis. Identification of such events provides important information for ongoing research on factors regulating the growth and differentiated function of steroidogenic cells. In one particular event, the cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450.sub.scc) controls the rate-limiting step of steroidogenesis, the enzymatic conversion of cholesterol to pregnenolone.
In one attempt to characterize the side-chain cleavage event, the amount of P-450.sub.scc has been investigated by immunofluorescent staining of P-450.sub.scc with an antibody to the P-450.sub.scc enzyme. See N. B. Goldring et al., "Immunofluorescent Probing of the Mitochondrial Cholesterol Side-Chain Cleavage Cytochrome P-450 Expressed in Differentiating Granulosa Cells in Culture,"119 Endocrinology, pp. 2821-2832 (1986). This technique, however, can not measure P-450.sub.scc enzyme activity since the immunofIuorescent staining requires cell fixation.
One measure of the cleavage event is the amount of the side chain fragment produced by the enzymatic cleavage. The enzymatic conversion of cholesterol to pregnenolone by the P-450.sub.scc enzyme can be measured from the amount of radioisotopically labeled isocapraldehyde formed from cholesterol when the sterol substrate bears a radioisotope on the side chain. See R. B. Hochberg et al., "A Simple and Precise Assay of the Enzymatic Conversion of Cholesterol in Pregnenolone,"13 Biochemistry, No. 13, pp. 603-608 (1974).
It would be desirable, however, to use a rapid, sensitive, single cell analysis technique, such as fluorescence detection in flow cytometry, for the study of these metabolic events. However, fluorescent probes have not been available that are specific to a particular cell function, such as the activity of a key enzyme. This problem is addressed by the present invention and a fluorescent probe is developed that is specific for identification of the rate-limiting cholesterol side-chain cleavage by the P-450.sub.scc enzyme.
Accordingly, it is an object of the present invention to provide a fluorescent probe to measure the activity of the enzyme responsible for the conversion of cholesterol to other steroids.
It is another object of the present invention to provide a steroid probe that is non-fluorescent until cleaved by P-450.sub.scc.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.